Factor VII is an important protein in the blood coagulation cascade. It is a vitamin K-dependent plasma protein that is synthesized in the liver and secreted into the blood as a single-chain glycoprotein, with a molecular weight of around 53 kDa. Synthesised as a zymogen, Factor VII is then converted into its activated form (FVIIa) by proteolytic cleavage at a single site, R152-I153, resulting in two chains linked by a single disulfide bridge. Recombinant human FVIIa is used for the treatment of bleeding episodes, e.g. in hemophilia or trauma and conditions associated with Factor VII/Factor VIIa deficiencies.
Purification of Factor VII (rFVII) or activated Factor VII (rFVIIa) is generally carried out using a combination of ion exchange and immuno-affinity chromatography that uses a calcium-dependant anti-FVII monoclonal antibody. Although this immuno-affinity based purification step is highly selective and provides protein of high purity, there are disadvantages. For example, the antibodies can potential leach into the final therapeutic product, which may affect the safety of the final composition. The cost of producing the monoclonal antibody (mAb) immuno-affinity matrix is considerably greater as compared to more conventional, non-antibody based purification matrices.
While replacement of the immuno-affinity step with a different purification technique would be advantageous, this would require removal of non-Factor VII-related contaminants, as well as the ability to separate any unwanted isoforms of Factor VII that may be present in the culture supernatants, such as aggregates. Examples of non-Factor VII-related cont74aminants include blood-borne products such as prothrombin, plasminogen, tissue plasminogen activator (tPA) and other proteases (e.g., where Factor VII is being purified from a blood product such as plasma) or it may include material derived from cell culture such as cell debris and cell culture media (e.g., where the Factor VII is recombinantly produced). The level of contaminants can adversely affect the final preparation and thus limit its use, particularly for human applications.
Other methods for the purification of Factor VII have been described. For example, US 20040063187 is directed to a method of purifying the proenzyme form of Factor VII using anion-exchange chromatography (Mono Q Sepharose resin). The solution containing the proenzyme form of Factor VII was added to the resin using a buffer solution of 20 mM Na acetate, 0.1 M glycine, pH 4.5. The fraction passing through was discarded and the bound proteins were eluted using 20 mM Na acetate, 2 M NaCl, pH 4.5. The eluate was diluted in a buffer of 5 mM Na citrate, 50 mM NaCl, pH 6.0.
US 20090047723 is directed to a method for purifying Factor VII (rFVII) or activated Factor VII (rFVIIa) by subjecting the proteins to liquid chromatography on a hydroxyapatite (HAP) column. A solution containing Factor VII was added to a hydroxyapatite column in 25 mM imidazole, approx. 6 mM NaCl, approx. 18 mM CaCl2, pH 6.5. The bound protein was washed first in 25 mM imidazole, pH 6.5, then using 100 mM Na-Phosphate, pH 6.3, followed by 150 mM Na-Phosphate, 1 M NaCl to elute contaminants and unwanted Factor VII isoforms. Factor VII protein was then eluted using Na-Phosphate, 1 M NaCl, with a Na-Phosphate concentration gradient of from 150 mM to 500 mM together with a pH gradient of from 6.3 to 8.0.
The present invention provides an improved method of purifying Factor VII and/or Factor VIIa from a solution containing either or both proteins by using mixed-mode (multi-modal) anion exchange chromatography. Whilst the method can be used to remove any non-Factor VII-related contaminant from the solution, it is particularly useful for removing solvent and/or detergent from the solution that has been introduced, for example, during a virus inactivation step.